摘要
采用原代培养的方法,从2.5月成年大鼠的嗅球分离培养嗅神经鞘细胞(OECs),培养6天后,用阿糖胞昔(Ara-C)抑制,差速贴壁,Forskolin和BPE营养物质处理。根据P75蛋白免疫细胞化学染色和形态学特征分析了所得细胞的纯度。同时对不同培养时期的OECs的形态进行观察和纯化后的活力测定。实验结果显示:(1)这种纯化方法简单,经济,快捷,所得的OECs纯度可达95％以上,并且随培养时间延长,细胞仍保持较高的纯度。(2)在培养早期2天到5天主要以巨噬细胞状、多极状、不规则状为主,培养中期7天到20天主要以扁平的双极、三极为主。晚期20天以后呈现双极、三极形态,其突起上有许多细小的棘突。(3)其中以培养早中期细胞的活力较好,培养20天以后,细胞活力较差。本研究为以OECs作为移植材料对促进神经再生的研究获得丰富的细胞来源奠定了基础。
Primarily cultured olfactory ensheathing cells were dissociated from 2. 5 month old rat. After being cultured 6 days in vitro , purification of olfactory ensheathing cells(OECs)was developed with cytosine arabinosideC Ara-C) treatment and differential adhesion method. OECs were stimulated to propagate by BPE and forskolin. Then the purity of OECs was determined according to immunostaining positively for P75 at different time. At the same time, OECs morphological feature was observed performed under phase contrast microscope, and properties of OECS in vitro were analysed. Furthermore, viability of OECs at distinct time in vitro after purification was assessed by MTT assays. The results demonstrated that (1) More than 95 % cells are OECs in this culture system, and highly enriched population of OECs still keep with prolongation of culture time(2) In 2 to 5 days in vitro, Cells exhibit three distinct morphological features, which are macrophage-like, multipolar and irregular-shaped. In 5 to 8 days of cuture, the majority of cells show flat bipolar and tripolar. When being cultured over 20 days,mainly the bipolar and multipolar cells remain in the culture, Which processes contain abundant tiny spines. (3) At the day of 2 to 20 in vitro,OECs possess high viability, and the viability decrease with the culture time. This experiment laid a foundation for studying the promotion of neural regeneration using OECs as a candidate.
出处
《细胞生物学杂志》
CSCD
北大核心
2001年第1期43-47,共5页
Chinese Journal of Cell Biology
基金
国家"973"课题资助项目
